Study of protein interactions is central to an understanding of the biological roles of gene products in vivo. There are numerous ways of analysing or dissecting polypeptide interactions, and one of the most powerful is by use of peptide aptamers and study of their behaviour. Peptides and peptide aptamers may be used free in solution. However, small peptides when unconstrained will tend to form structures which present a limited interaction surface. Furthermore, they will often lose conformational entropy upon association with target molecules, reducing free energy of binding and consequently free peptides will often not form tight non-covalent complexes, which is a problem.
Rather than being used in free solutions, peptides of interest may be bound to physical supports, or displayed in the context of a larger polypeptide. It is display in the context of a polypeptide which is important in the present invention. Such display is often brought about using scaffold proteins.
Engineered protein scaffolds for molecular recognition have been produced and used in the prior art. For example, Skerra (2003 Curr Opin Chem Biol. vol. 7 pages 683-93) discusses scaffolds used for the generation of artificial receptor proteins with defined specificites. According to Skerra, the best scaffolds should have robust architecture, small size, be monomeric, be susceptible to protein engineering (eg. fusion proteins) and have a low degree of post-translational modification. Furthermore, the most advantageous scaffolds should be easy to express in host cells (usually prokaryotic cells in the prior art), have a region susceptible to insertion or replacement of amino acids to create novel binding sites, and such insertion/replacement of binding sites should not affect folding of the scaffold.
The most commonly used scaffolds are based on the framework regions of immunoglobulin or ‘antibody’ chains. In particular, the Ig framework and/or shortened or fused versions of it have been used to present and geometrically constrain peptides in the prior art. However, antibodies are large, and even the recombinant fragments are of considerable size (eg. Fab fragments are about 450aa, and even scFv fragments are about 270aa). This makes them awkward to manipulate in vitro and in vivo. Furthermore, they are comprised of two different polypeptide chains which are unstable in the sense of dissociation, oligomerisation and even wholesale aggregation, which represent further problems associated with their use.
Prior art scaffolds have included inactivated staphylococcal nuclease, green fluorescent protein (GFP) and thioredoxin A (TrxA), as well as isolated protein folds such as the Z domain of staphylococcal protein A, “affibodies”, anticalins, and ankyrin repeats. Further prior art scaffold proteins include the fibronectin type III domain (‘Fn3’), lipocalin family proteins from which anticalins are derived, bilin binding protein (BBP), and others.
This technology has been most actively pursued using bacterial thioredoxin (TrxA) as a scaffold. However, there are problems associated with TrxA. For example, E. coli TrxA can inhibit apoptosis which may lead to confounding observations in cell-based assays. Also, the two cysteine residues which border inserted peptides, and which form a reversible disulphide bond in TrxA, can lead to uncertainty regarding the “correct” state for presentation of active peptide.
The present invention seeks to overcome problem(s) associated with the prior art.